The rational development of new antineoplastic agents directed against tubulin, a protein critical for cell division, requires greater understanding of the interactions between the polypeptide subunits of tubulin and its two tightly bound guanine nucleotides. Interactions of ribose-modified GDP and GTP analogs with tubulin were examined and suggested that there are actually two mutually exclusive exchangeable nucleotide binding sites. Antimitotic drugs were examined for their effects on tubulin-dependent GTP hydrolysis. The stimulatory effect of colchicine appears to derive from its trimethoxybenzene moiety. Nocodazole also stimulated GTP hydrolysis, while maytansine inhibited the reaction. Requirements for GTP and its hydrolysis were profoundly affected by other reaction components in the presence of taxol. Hydrophobic chromatography was successful in separating alpha-tubulin from beta-tubulin.